Combination antiretroviral therapy (ART) has resulted in a major decrease in morbidity and mortality in HIV- infection. However, ART does not eradicate virus or restore HIV-specific immune responses capable of controlling viral replication once therapy is interrupted. Development of interventions that could generate immune responses capable of maintaining HIV suppression after cessation of ART and improve efficacy of vaccine candidates would be a crucial clinical advance. Evidence suggests that effective HIV-specific CD4 T cells will be required to achieve this goal, yet these cells remain seriously understudied. The lack of restoration of effective HIV-specific CD4 T helper responses in spite of extended ART raised the possibility that these responses are irreversibly crippled. However, the results we have generated during the first four years of this grant support a striking conclusion: that CD4 T cell impairment in HIV-infection is to a significant extent under control of inhibitory mechanisms that can be reverted by manipulation of regulatory networks. Data suggest that one of these pathways, PD-1, also plays an important role in the maintenance of HIV reservoirs, making it a very attractive therapeutic target. In this competing renewal we propose to build on the major progress made during the initial funding period and to utilize combined approaches of cellular biology, molecular immunology and advanced cell imaging to pinpoint critical events that regulate effective and inefficient HIV-specific CD4 T cell responses. We also propose to investigate another neglected area of HIV-research: the impact that ineffective HIV-specific CD4 T cell responses have on antigen-presenting cell functions. In Aim 1, we will identify critical gene signatures of functional and exhausted HIV-specific CD4 T cells. We will determine the master transcription factors that are regulated by PD-1 in HIV-specific CD4 T cells and define their role in the control of exhausted CD4 T cell functions. We will examine the potential for HIV-specific CD4 T cell functional plasticity by overexpressing or silencing these master genes in primary cells. In Aim 2 we will use advanced live imaging to determine the dynamics of immunological synapses built by functional and exhausted HIV- specific CD4 T cells. We will define the role of individual co-inhibitors and determine whether optimized co- stimulatory signaling can prevent this defective synapse formation and function. In Aim 3 we will define specific defects in licensing of antigen-presenting cells by HIV-specific CD4 T cells, and specific mechanisms of APC impairment. We will determine whether APC present inappropriate responses to stimulatory signals. By selectively blocking molecules expressed by dysfunctional CD4 T cells, we will determine events causally involved in the perturbation of APC functions. We will also define how the interplay between regulatory pathways leads to the observed defects. This proposal will provide novel understanding of the molecular basis for functional differences between protective and ineffective HIV-specific CD4 T cells and will identify new therapeutic targets to generate effective HIV-specific immunity in ART-treated and uninfected persons.